Eukaryotic gene expression must be regulated such that cells can rapidly respond to a wide range of different conditions. The process of mRNA translation is one step at which gene expression is highly regulated. In response to hormones, growth factors, cytokines and nutrients, animal cells generally activate translation in preparation for the proliferative response. The rate of protein synthesis typically decreases under stressful conditions, such as oxidative or osmotic stress, DNA damage or nutrient withdrawal. Activation or suppression of mRNA translation occurs within minutes and control over this process is thought to be exerted at the initiation phase of protein synthesis (Rosenwald et al., Oncogene, 1999, 18, 2507-2517; Strudwick and Borden, Differentiation, 2002, 70, 10-22).
Translation initiation necessitates the coordinated activities of several eukaryotic initiation factors (eIFs), proteins which are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. One of these factors, eukaryotic initiation factor 4E (eIF4E), is present in limiting amounts relative to other initiation factors and is one component of the eIF4F initiation complex, which is also comprised of the scaffold protein eIF4G and the RNA helicase eIF4A. In the cytoplasm, eIF4E catalyzes the rate-limiting step of cap-dependent protein synthesis by specifically binding to the 5′ terminal 7-methyl GpppX cap structure present on nearly all mature cellular mRNAs, which serves to deliver the mRNAs to the eIF4F complex. Once bound, the eIF4F complex scans from the 5′ to the 3′ end of the cap, permitting the RNA helicase activity of eIF4A to resolve any secondary structure present in the 5′ untranslated region (UTR), thus revealing the translation initiation codon and facilitating ribosome loading onto the mRNA (Graff and Zimmer, Clin. Exp. Metastasis, 2003, 20, 265-273; Strudwick and Borden, Differentiation, 2002, 70, 10-22).
eIF4E availability for incorporation into the eIF4E complex is regulated through phosphorylation as well as through the binding of inhibitory proteins. eIF4E is a phosphoprotein that is phosphorylated on serine 209 by the mitogen-activated protein kinase-interacting kinase Mnk1, as well as by protein kinase C (Flynn and Proud, J. Biol. Chem., 1995, 270, 21684-21688; Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; Waskiewicz et al., Embo J., 1997, 16, 1909-1920). The inhibitory eIF4E-binding proteins 1 and 2 (eIF4E-BP1 and eIF4E-BP2) act as effective inhibitors of cap-dependent translation by competing with eIF4G for binding to the dorsal surface of eIF4E (Pause et al., Nature, 1994, 371, 762-767; Ptushkina et al., Embo J., 1999, 18, 4068-4075). When complexed with bp1, eIF4E is not a substrate for phosphorylation by protein kinase C or Mnk1, indicating that dissociation of bp 1 from eIF4E is a prerequisite for eIF4E phosphorylation (Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; Whalen et al., J Biol Chem, 1996, 271, 11831-11837). Phosphorylation of eIF4E increases its affinity for mRNA caps, thus elevating translation rates (Waskiewicz et al., Mol. Cell. Biol., 1999, 19, 1871-1880).
eIF4E-BP2 (also known as PHAS-II; 4EBP2; 4E-binding protein 2; EIF4EBP2) was cloned through use of the eIF4E protein in probing a cDNA expression library (Hu et al., Proc Natl Acad Sci U S A, 1994, 91, 3730-3734; Pause et al., Nature, 1994, 371, 762-767). eIF4E-BP2 is ubiquitously expressed in human tissues, including heart, brain, placenta, lung, liver, kidney and spleen, as well as adipose tissue and skeletal muscle, the major insulin-responsive tissues (Hu et al., Proc Natl Acad Sci U S A, 1994, 91, 3730-3734; Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The human gene maps to chromosome 10q21-q22 (Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The mouse bp1 gene consists of three exons, spans approximately 20 kb and maps to mouse chromosome 10 (Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The expression of eIF4E-BP2 does not appear to be altered in mice bearing a systemic disruption of bp1 (Blackshear et al., J Biol Chem, 1997, 272, 31510-31514).
Rather than preventing the binding of eIF4E to mRNA caps, eIF4E-BP2 prohibits the binding of eIF4E to eIF4G, thereby preventing formation of a complex that is necessary for efficient binding and proper positioning of the 40S ribosomal subunit on the target mRNA. When eIF4E-BP2 is bound to eIF4E, eIF4E does not serve as a substrate for phosphorylation by protein kinase C, suggesting that dissociation of eIF4E-BP2 from eIF4E is a prerequisite for phosphorylation of eIF4E (Whalen et al., J Biol Chem, 1996, 271, 11831-11837). The region to which eIF4E binds is a common motif shared by eIF4G and eIF4E-BP2, and point mutations in this region of eIF4E-BP2 abolish binding to eIF4E (Mader et al., Mol Cell Biol 1995, 15, 4990-4997). Two conserved motifs are present in the eIF4E-BP2: the RAIP motif, which is found in the NH2-terminal region of EIF4E-BP2 and the TOS motif, which is formed by the last five amino acids of eIF4E-BP2 (Schalm and Blenis, Curr Biol, 2002, 12, 632-639; Tee and Proud, Mol Cell Biol, 2002, 22, 1674-1683).
Like eIF4E-BP1, insulin stimulates the phosphorylation of eIF4E-BP2 in cultured cells, which promotes the release of eIF4E-BP2 from eIF4E and allows for cap-dependent translation to proceed Ferguson et al., J Biol Chem, 2003, 278, 47459-47465). Mitogen-activated protein kinase, the major insulin-stimulated kinase in rat adipocytes, can phosphorylate recombinant eIF4E-BP2 in vitro. However, treatment of 3T3-L1 rat adipocytes with rapamycin attenuates the effects of insulin on the phosphorylation of eIF4E-BP2, indicating that elements of the mTOR signaling pathway mediate the actions of insulin on eIF4E-BP2 (Lin and Lawrence, J Biol Chem, 1996, 271, 30199-30204). Additionally, serine-65 of eIF4E-BP2 represents an ideal consensus site for phosphorylation by cyclicAMP-dependent protein kinase. In rat 3T3-L1 adipocytes, where insulin or epidermal growth factor markedly increased the phosphorylation of eIF4E-BP2, compounds that increase cyclic AMP decrease the amount of radiolabeled phosphate incorporated into eIF4E-BP2, and attenuate the effects of insulin on increasing the phosphorylation of eIF4E-BP2. Incubation of eIF4E-BP2 with the catalytic subunit of cyclic AMP-dependent protein kinase results in the rapid phosphorylation of eIF4E-BP2. Together, these data suggest that increasing cyclic AMP may selectively increase eIF4E-BP2 phosphorylation (Lin and Lawrence, J Biol Chem, 1996, 271, 30199-30204).
Induction of cellular differentiation and reduction of cellular proliferation are concomitant with a reduction in translation rates, as is observed in conduction with differential regulation of eIF4E-BPs during human myeloid cell differentiation. When induced to differentiate into monocytes/macrophages, cells from the HL-60 promyelocytic leukemia cell or U-937 monoblastic cell lines exhibit a decrease in the phosphorylation of bp1. In contrast, when HL-60 cells are stimulated to differentiate into granulocytic cells, the amount of bp1 is decreased, whereas phosphorylation of bp1 is not affected. Conversely, eIF4E-BP2 levels are markedly increased. These findings suggest that translation machinery is differentially regulated during human myeloid cell differentiation (Grolleau et al., J Immunol, 1999, 162, 3491-3497).
The disregulation of signaling networks that promote cell proliferation is often observed in association with cancer (Lawrence and Abraham, Trends Biochem Sci, 1997, 22, 345-349). Expression of excess eIF4E-BP2 in cells transformed by eIF4E or v-src results in significant reversion of the transformed phenotype, demonstrating that eIF4E-BP2 can function as an inhibitor of cell growth (Rousseau et al., Oncogene, 1996, 13, 2415-2420).
The U.S. Pat. No. 6,410,715 describes a purified human nucleic acid sequence encoding a cellular component that binds to eIF4E comprising a coding sequence for the protein eIF4E-BP2, and discloses a method for screening a non-hormone agent potentially useful to treat a hormone disorder (Sonenberg et al., 2000).
Currently, there are no known therapeutic agents that target eIF4E-BP2. Consequently, there remains a long felt need for agents capable of effectively inhibiting eIF4E-BP2. Antisense technology is an effective means of reducing the expression of specific gene products and therefore is uniquely useful in a number of therapeutic, diagnostic and research applications for the modulation of eIF4E-BP2 expression.
The present invention provides compositions and methods for inhibiting eIF4E-BP2 expression.